Development of efficacious and robust biologics is a complex process. Light scattering technologies assist at each stage with uniquely versatile technologies for biophysical screening and characterization, from target and candidate discovery to selection, optimization, purification, formulation and manufacturing scale-up.
Some key applications of light scattering in biotherapeutic development are described below.
Target & candidate proteins
Once target proteins and candidate biomolecules for a given indication have been identified, they must be produced at a small scale in sufficient quantities and appropriate quality for further R&D.
Size exclusion chromatography with multi-angle light scattering (SEC-MALS) accurately characterizes proteins and other biomolecules for soluble aggregates, regardless of non-ideal column interactions, to quickly identify optimal purification conditions.
High-throughput dynamic light scattering (DLS) using the DynaPro® Plate Reader requires minimal sample quantities and time to assess candidates and pre-formulation conditions for stability and propensity for aggregation.
Composition-gradient, multi-angle light scattering (CG-MALS) combining a Calypso composition-gradient system and DAWN® MALS detector determines affinity and absolute stoichiometry of drug-target binding, without labeling or immobilization. It is particularly well suited for studying multivalent interactions or drug-target binding in the presence of self-association, including virus-like particles (VLPs), monomeric antibodies, oligonucleotides or peptides.
Candidate selection & optimization
Post-translational modifications such as glycosylation or PEGylation are often applied to proteins or peptides in order to improve stability and increase physiological retention time. Some biologics, such as insulin, are required to form well-defined oligomeric states for the same reason. Modification of IgG with conjugated, highly toxic drugs can convert an antibody to a potent anti-cancer substance.
Triple-detection SEC-MALS utilizing combined UV, MALS and RI instruments readily determines the degree of glycosylation or PEGylation, and in many instances may be used to calculate the drug-antibody ratio (DAR) of antibody drug conjugates (ADCs).
CG-MALS analyzes reversible oligomerization by measuring the dependence of weight-average molar mass on concentration. The oligomeric order may be determined unambiguously, along with the affinity.
Antibody engineering and other modifications by means of mutations to the genetic sequence may be carried out in order to improve stability or efficacy. SEC-MALS helps determine the impact of these changes on aggregation, while CG-MALS assesses how they have affected target binding properties.
For larger entities such as virus-like particles (VLPs) and polymer, liposome or hydrogel-based drug carriers, separation by field-flow fractionation (FFF) is usually preferable to size exclusion chromatography. The Eclipse™ DualTec™ and Eclipse AF4 FFF systems offer a variety of options for optimal separation, upstream of a MALS detector.
Light scattering measurements help process development in several ways:
Purification end point
Since multi-angle light scattering determines molar masses independently of retention time, RT-MALS is ideal for real-time characterization of purification products and determination of end points. For more detailed product characterization during process development, SEC-MALS may be implemented at-line with UHPLC for rapid separation and analysis.
Complex products such as virus-like particles or peptide micelles must be produced with specific masses, shapes and sizes, as well as minimum quantities of residual unassembled material. SEC-MALS or FFF-MALS with on-line DLS validates such processes to quantify unassembled sub-units and ensure that the final product conforms to the required structure.
In the formulation process, many combinations of buffer conditions and excipients must be tested for stability and propensity for aggregation. Final selection of the most promising conditions for long-term testing does not necessarily require rigorous thermodynamic characterization, but does benefit from an understanding of multiple properties which reflect various facets of stability. High-throughput DLS is the ideal technique for this, as it can address multiple stability-indicating parameters simultaneously testing thousands of conditions per day:
- Aggregation state - formation of aggregates, size distribution and rate of formation. This is the basic DLS analysis.
- Thermal stability - how temperature induces denaturing and non-native aggregation. Determined through temperature ramps, monitoring changes in molecular size.
- Chemical stability - how buffer composition (pH, ionic strength, denaturing solvents such as guanidine) induces denaturing and non-native aggregation. Determined through composition steps, monitoring changes in molecular size.
- Colloidal stability - how surface moieties such as charged residues or hydrophobic patches lead to aggregation, unrelated to changes in tertiary structure. Determined through the concentration dependence of translational diffusion, kD or D1.
With the DynaPro Plate Reader, these measurements takes place in situ in standard 96, 384 or 1536 microwell plates, consuming minimal sample quantities. The same samples may be further tested in the same or other plate-based instruments [e.g., to analyze the temperature dependence of colloidal stability (in the DynaPro) or intrinsic fluorescence (in a spectroscopic plate reader)], making the DynaPro Plate Reader an invaluable tool for formulation screening. A software package enables integration of the instrument with robotic sample preparation and plate placement.
Another helpful light scattering tool for formulation development is Electrophoretic Light Scattering (ELS). The Mobius™ integrates with an autosampler and the Atlas for automated measurement of molecular charge, an important parameter for stability. The measurement takes place in native buffers, even under moderate to high salt conditions. Like the DynaPro® Plate Reader, the Mobius offers automated measurement of multiple parameters such as thermal and colloidal stability.
Additional characterization challenges in the course of high-concentration formulation of monoclonal antibodies and other biotherapeutics include understanding the interplay of weak interactions, and viscosity.
Weak protein-protein interactions may not be important at low concentrations, and so are not necessarily reflected in dilute-condition parameters such as kD, but come to dominate at high concentration. CG-MALS uniquely addresses these complex interactions through a theoretical framework known as the Minton-Chatelier formalism. This technique distinguishes attractive interactions causing aggregation and viscosity, from repulsive interactions which may impact measurements but have little effect on stability.
Viscosity is both indicative of colloidal stability, and an important parameter on its own since high viscosity impacts manufacturability and deliverability of the final product. DLS readily determines viscosity of concentrated protein solutions using tracer beads. The DynaPro Plate Reader can evaluate the viscosity of samples as small as 10 µL in a few seconds, measuring thousands of conditions per day in order to screen for ideal viscosity-reducing excipients.
Light scattering is an essential part of any biophysical characterization lab for biotherapeutics, and is recognized by the FDA as a key technology for regulatory filings. Key applications include:
Applying orthogonal techniques based on light scattering and other instruments provided by Wyatt, analytical support labs can support regulatory filings with thoroughly validated, extended analysis of soluble and insoluble sub-micron aggregates. These techniques are:
- SEC-MALS for robust characterization of the mass and size distributions of soluble, irreversible aggregates
- FFF-MALS for robust characterization the mass and size distributions of soluble and insoluble, irreversible aggregates
- Batch DLS for rapid testing of size distributions for soluble and insoluble, reversible and irreversible aggregates
- CG-MALS for characterization of reversible aggregates or oligomers, as well as validation of SEC-MALS for loss of aggregates on the column.
Triple detection by UV-MALS-dRI determines the composition as well as molar mass distributions of:
- Protein-polysaccharide conjugated vaccines
- PEGylated proteins
- Antibody-drug conjugates
- VLPs with DNA/RNA payloads
With the addition of a WyattQELS DLS module, the size distributions may be determined as well.
- Chemical disposition: ELS determines charge state and so is an important indicator of surface chemistry.
- Functionality: CG-MALS is a powerful technique for in-depth characterization of biomolecular interactions such as drug-target binding, without labeling or immobilization. Since it measures molar mass directly, it is particularly versatile for a range of valencies and self- as well as hetero-association.
The large variety of components utilized in different kinds of vaccines make it difficult to summarize all the ways in which light scattering are applied to vaccine development. Here are a few:
Polysaccharides tend to be very heterogeneous; properties of interest include distributions of molar mass and size as well as conformation. In most instances SEC-MALS combined with online DLS provides good resolution and complete information. When the polysaccharide molar masses are very high and the molecules highly branched, FFF-MALS-DLS will provide better data since the 'anchoring' effect causing anomalous SEC separation does not occur in FFF.
SEC column calibration cannot be used for characterizing the molecular weights of protein-polysaccharide conjugates, because relevant standards do not exist. Only SEC-MALS employing the triple-detector technique (UV-MALS-RI) and 'Conjugate Analysis' algorithms provides accurate and reliable analysis of molar masses, providing not only the total molecular weight but also the masses of the constituent protein and polysaccharide. Even if an additional component such as lipid is incorporated into the complex, it is possible to determine protein and total molar masses.
The same analysis may be applied to FFF separations.
Viruses and virus-like particles (VLP)
Light scattering coupled to FFF is one of the more convenient methods for determining virus particle counts, with results very similar to those of cryo-TEM.
DLS, SEC-MALS and FFF-MALS are used extensively in the course of developing and producing VLPs:
- SEC-MALS for assessing assembly of capsomeres into well-formed VPS as well as VLP molecular weight, size, aggregation state, shape and even payload
- FFF-MALS for the same characterizations when VLPs do not elute well from SEC columns
- DLS for high-throughput formulation
Lipsosomes may be used as adjuvants as well as delivery vehicles for antigens. DLS is an excellent means of rapidly assessing liposome size. When more accurate distributions are needed or additional characterization such as shape or payload, SEC-MALS and FFF-MALS provide reliable results with excellent resolution.
Need to assess the degree antibody binding to an isolated antigen protein or a complex vaccine such as a VLP? CG-MALS determines binding affinity and absolute molecular stoichiometry, in solution, without labeling. It is also invaluable in assessing protein-protein interactions in multi-valent vaccines that include more than one antigen.
In current biotherapeutic manufacturing strategies, “the process is the product” means that strict control of process parameters such as pH, temperature, and feedstock consumption is required to guarantee product quality. Only at the end of the process can QC procedures check the critical quality attributes (CQAs) that define product quality. Hence, most PAT instruments monitor process conditions rather than product attributes.
Real-time multi-angle light scattering (RT-MALS) is a novel PAT application of light scattering, that directly determines the molecular weight of mAbs and other therapeutic biomolecules. It is particularly sensitive to the appearance of aggregates and contaminant particles. Using the ultraDAWN™ RT-MALS instrument, in-line or on-line with purification and other process steps, production staff can speed up and optimize process development with immediate feedback on the impact of process variations and deviations. UltraDAWN measures another attribute, the average radius, for virus-like particles and other biomolecular complexes with diameters above 25-30 nm.
After developing the process, the ultraDAWN may be left on-line. Its accompanying software, OBSERVER™, integrates with an OPC-AU environment for automated and/or GMP operation. OBSERVER can be programmed to trigger the system to react to deviations from the allowed molar mass range due to aggregation, or to shut down a reaction once the endpoint has been reached.
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